Targeting the HIV entry and assembly pathways holds promise for development of anti- HIV gene therapy vectors as disrupting the virus at multiple stages in the life cycle will likely limit the emergence of resistant mutants. Moreover, recent advances in human CD34+ stem cell transduction and transplantation have paved a way for anti-HIV gene therapy in the near future. These advances have thus prompted a search for new and potent gene therapy targets for suppression of HIV replication. While si/shRNAs against viral and cellular factors have been tested there are a limited number of studies on dominant negative (DN) viral or cellular proteins that can act as HIV inhibitors. In this regard, the Gag and Envelope proteins of HIV-1 remain an attractive yet unexploited target for anti-HIV gene therapy. Both the Gag and envelope proteins play essential roles in completion of the viral life cycle namely via promoting HIV particle assembly/budding or allowing infection into cells respectively. Notably, virus assembly not only requires the viral Gag protein but also numerous host cell factors like Tsg101, Alix, GGAs, Arfs, POSH, AP-1, AP-3 proteins etc to complete virion morphogenesis. Hence, dominant negative forms of the above genes either individually or in combination have the potential to be developed as powerful tools to target HIV-1 replication. Thus, targeting HIV replication via multiple ways will have the advantage of not only halting virus spread rapidly but also restrict the emergence of resistant isolates. PUBLIC HEALTH RELEVANCE: The rapidly mutating nature of HIV and the adverse effects associated with routine drug therapy solicits the development of alternative therapeutic interventions. One of the obvious alternatives to chemotherapy is gene therapy. Recent advances in genetic manipulation of CD34+ stem cells, along with the development of lentiviral vectors capable of delivering desired genes to non-dividing cells has made HIV-based gene therapy a realistic possibility. An attractive gene therapy approach is to utilize dominant negative (DN) HIV proteins to target HIV life cycle. In this regard the Gag and Envelope (Env) proteins of HIV-1 remain an attractive yet unexploited target because both the Gag and Env proteins are essential components of the virus life cycle and the use of dominant negative forms of these genes individually or in combination have not been thoroughly investigated for anti-HIV gene therapy. In this proposal we aim to identify and design viral vectors expressing DN Gag and Env proteins individually and in combination and test their potential to inhibit WT HIV replication. We also propose to determine whether host factors important for HIV-1 assembly and release like Tsg101 and Alix can be targeted in cells by expression of their DN counterparts and developed as novel gene therapy targets. This approach will not only help identify novel therapeutic targets for HIV intervention but also help decipher the role of known and unknown host factors in the virus replication cycle.